Effective replication of human influenza viruses in mice lacking a major alpha2,6 sialyltransferase

The hemagglutinins of influenza viruses isolated from humans typically prefer binding to sialic acid in an alpha2,6 linkage. Presumably, the virus uses the presence of these receptors on the respiratory tract to gain entrance into the host cell. The ST6Gal I sialyltransferase knock-out mouse lacks the main enzyme necessary for the attachment of alpha2,6 sialic acid to N-linked glycoproteins on the cell surface. Yet even in the absence of detectable alpha2,6 sialic acid in the mouse respiratory tract, human influenza viruses can still infect these mice and grow to similar titers in the lung and trachea as compared to wild-type animals. This work demonstrates that the presence of a major alpha2,6 sialic acid on N-linked glycoproteins is not essential for human influenza virus infection in mice.     

Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kit gene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.     

Metabolic and physiological differences between zero-flow and low-flow myocardial ischemia: effects of L-acetylcarnitine

Summary The metabolic and physiologic differences between low-flow and zero-flow ischemia of varying duration were compared in the isolated perfused rat heart. Hearts subjected to 60 and 90 minutes of zero-flow ischemia recovered less cardiac work than hearts subjected to low-flow ischemia. Low-flow ischemia caused a build-up of both myocardial long-chain acyl coenzyme A and acyl carnitine esters, while zero-flow ischemia produced no change in long-chain acyl carnitine and only a transient increase in long-chain acyl coenzyme A. High energy phosphate depletion was greater in zero-flow ischemia. Perfusion with excess free fatty acids decreased the recovery of cardiac work after low-flow ischemia but had no effect after repeated episodes of zero-flow ischemia. L-Acetylcarnitine improved the recovery of cardiac work after low-flow ischemia in hearts perfused with 0.4 and 1.2 mM palmitate. With zero-flow ischemia, L-acetylcarnitine had no effect on the recovery of cardiac work in hearts perfused with 0.4 mM palmitate and a slight but statistically significant effect with 1.2 mM palmitate. Possible protective mechanisms of L-acetylcarnitine against ischemic damage are discussed.     

Beyond survival: how well do transplanted livers work? A preliminary comparison of standard‐risk,high‐risk,and living donor recipients

To help decrease mortality on the liver transplant waitlist, transplant centers are using living donors (LD) and high‐risk donors (HRD) in addition to standard‐risk donors (SRD). HRD is defined as having a donor risk index score higher than 1.6, which suggests a great risk of graft failure. Recent studies have examined survival rates between HRD and SRD recipients; however, little is known about outcomes other than survival, specifically psychosocial outcomes. The purpose of this preliminary, prospective study was to compare post‐transplant psychosocial and recovery outcomes between SRD and LD and HRD liver recipients. These outcomes include cognitive functioning, psychological distress, quality of life, and self‐reported and objective measures of recovery. Eighty‐four patients provided baseline and six‐month post‐transplant data. There were generally no statistically significant differences at baseline or the six‐month follow‐up, suggesting that patients receiving HRD livers have similar outcomes to those who receive SRD livers. However, some effect sizes suggest potential advantages for LD recipients compared to SRD recipients. Transplant centers may be more willing to encourage patients to accept HRD or LD livers knowing that they may have comparable outcomes to SRD recipients, which also has implications for the transplant waitlist.     

Prediction of peak oxygen uptake from differentiated ratings of perceived exertion during wheelchair propulsion in trained wheelchair sportspersons

Purpose

To assess the validity of predicting peak oxygen uptake ( $ {\dot{\text{V}}}{\text{O}}_{{\text{2peak}}}$ ) from differentiated ratings of perceived exertion (RPE) obtained during submaximal wheelchair propulsion.

Methods

Three subgroups of elite male wheelchair athletes [nine tetraplegics (TETRA), nine paraplegics (PARA), eight athletes without spinal cord injury (NON-SCI)] performed an incremental speed exercise test followed by graded exercise to exhaustion ( $ {\dot{\text{V}}}{\text{O}}_{{\text{2peak}}}$ test). Oxygen uptake ( $ {\dot{\text{V}}}{\text{O}}_2$ ), heart rate (HR) and differentiated RPE (Central RPE_C, Peripheral RPE_P and Overall RPE_O) were obtained for each stage. The regression lines for the perceptual ranges 9–15 on the Borg 6–20 scale ratings were performed to predict $ {\dot{\text{V}}}{\text{O}}_{{\text{2peak}}}$ .

Results

There were no significant within-group mean differences between measured $ {\dot{\text{V}}}{\text{O}}_{{\text{2peak}}}$ (mean 1.50 ± 0.39, 2.74 ± 0.48, 3.75 ± 0.33 L min^?1 for TETRA, PARA and NON-SCI, respectively) and predicted $ {\dot{\text{V}}}{\text{O}}_{{\text{2peak}}}$ determined using HR or differentiated RPEs for any group (P > 0.05). However, the coefficients of variation (CV %) between measured and predicted $ {\dot{\text{V}}}{\text{O}}_{{\text{2peak}}}$ using HR showed high variability for all groups (14.3, 15.9 and 9.7 %, respectively). The typical error ranged from 0.14 to 0.68 L min^?1 and the CV % between measured and predicted $ {\dot{\text{V}}}{\text{O}}_{{\text{2peak}}}$ using differentiated RPE was ≤11.1 % for TETRA, ≤7.5 % for PARA and ≤20.2 % for NON-SCI.

Conclusions

Results suggest that differentiated RPE may be used cautiously for TETRA and PARA athletes when predicting $ {\dot{\text{V}}}{\text{O}}_{{\text{2peak}}}$ across the perceptual range of 9–15. However, predicting $ {\dot{\text{V}}}{\text{O}}_{{\text{2peak}}}$ is not recommended for the NON-SCI athletes due to the large CV %s (16.8, 20.2 and 18.0 %; RPE_C, RPE_P and RPE_O, respectively).     

Blood-brain barrier permeability in galactosamine-induced hepatic encephalopathy. No evidence for increased GABA-transport

Blood-brain barrier permeability to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), to sucrose and to sodium was studied in rats with galactosamine-induced liver damage and hepatic encephalopathy by means of an arterial integral uptake technique. Permeability to GABA was unaltered in all examined brain regions (2.47 +/- 0.25.10(-5) cm3.s-1.g-1, mean +/- S.D.) as compared to control rats (2.49 +/- 0.19.10(-5) cm3.s-1.g-1). The permeability to sucrose (galactosamine 0.25 +/- 0.02 vs. controls 0.24 +/- 0.02.10(-5) cm3.s-1.g-1) and to sodium (galactosamine 5.33 +/- 0.04 vs. controls 5.40 +/- 0.05.10(-5) cm3.s-1.g-1) was also unchanged in hepatic encephalopathy. At the time of investigation mean liver function measured by antipyrine clearance was reduced from 0.39 in control rats to 0.23 ml/min/100 g body wt. in galactosamine-treated animals. The present study does not support the suggestion that peripheral GABA penetrates the blood-brain barrier to any higher extent in hepatic encephalopathy. This provides evidence against at least part of the GABA-hypothesis. Furthermore, an unspecific increased blood-brain barrier permeability in hepatic encephalopathy, as measured by sucrose and sodium uptake, was not found. It is concluded that the GABA-theory requires further careful reevaluation.     

Probing the binding specificities of human Siglecs by cell-based glycan arrays

Siglecs are a family of sialic acid–binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2–3(6-O-sulfo)Galβ1–4GlcNAc (6′-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer’s disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid–binding proteins.

Immune cells are equipped with an array of glycan-binding proteins (GPBs) capable of interpreting the biological information encoded by glycans. Endogenous GBPs recognize host-derived “self” and foreign-derived “nonself” glycans and produce cues that are integrated into the signaling network of immune cells and contribute to immune homeostasis and the immune response (1). Siglecs (sialic acid–binding immunoglobulin-like lectins) serve in self-recognition and transmit immune inhibitory signals upon binding to a select repertoire of sialoglycans expressed by host cells raising the threshold for immune activation (2, 3). The human Siglec family consists of 14 functionally expressed members, and these are composed of an N-terminal V-set immunoglobulin (Ig)-like domain that mediates sialoglycan binding followed by varying numbers of C2-set Ig-like domains. Intracellularly, most Siglecs have immunoreceptor tyrosine-based inhibition motifs, and Siglec-14/15/16 carry immunoreceptor tyrosine-based activation motifs (3–7). Siglecs are broadly expressed throughout the immune system, and several Siglecs are also found outside of the immune system, such as Siglec-4 (MAG), which is expressed by oligodendrocytes and Schwann cells in the nervous system (8). Although the diverse biological functions within and outside of the immune system of Siglecs are not fully understood, Siglecs generally contribute to immune homeostasis by dampening immune activation upon recognition of sialoglycans. For example, Siglec-2 (CD22) can suppress B cell receptor activation (9), and Siglec-9 can dampen neutrophil activation (10). Cancer cells with aberrant sialoglycans and pathogens that express sialic acids can exploit Siglec signaling to modulate immune responses (11, 12). Moreover, Siglec-3 (CD33) is strongly associated with risk for Alzheimer’s disease and expressed on microglia cells (13, 14). Given the potent immune modulatory functions of Siglecs and their wide involvement in autoimmunity, infection, cancer, and neurodegeneration, Siglecs are promising therapeutic targets (7, 15). However, many of the natural ligands of Siglecs have not been fully identified, and endogenous ligands for several Siglecs including Siglec-3/CD33 remain elusive.Human cells can produce a large diversity of glycans capped with sialic acids (Sia), a family of chemically diverse sugars with N-acetylneuraminic acid (Neu5Ac) being the predominant type in humans. Sialic acids are generally found at the termini of mammalian glycans, and most types of glycoconjugates including N-glycoproteins, multiple types of O-glycoproteins, and glycolipids carry oligosaccharides capped by sialic acids (16, 17). Sialylation is one of the most complex regulated steps in glycosylation with 20 distinct Golgi-located sialyltransferase isoenzymes dedicated to catalyze transfer of sialic acids to galactose (ST3GAL1-6, ST6GAL1 and 2), N-Acetylgalactosamine (Gal-NAc) (ST6GALNAC1-6), or sialic acid (ST8SIA1-6) via α2-3, α2-6, or α2-8 linkages, respectively and with different preferences for the underlying glycan structures and types of glycoconjugate (18–20). The resulting plethora of sialic acid-containing glycans constituting the sialome of cells provides a vast catalog of ligands for Siglecs and potential for distinct instructive cues for the immune response (16). The current insight into the interactome of Siglecs is largely derived from studies with libraries of synthetic and natural glycans printed on glass arrays (21, 22). These glycan arrays have demonstrated distinct structural glycan features that drive selective binding of individual Siglecs, including the linkage type of sialic acids, the core disaccharide carrying sialic acids, and glycan modifications such as sulfation or acetylation (23–27). However, printed glycan arrays may not present glycans in the natural context of the overall glycoconjugate structure and the cell surface with spatial organization and competition dynamics limiting insight into the fine binding specificities of Siglecs and their interactions with the host cell sialome.Here, we took advantage of our recently developed cell-based glycan array strategy (28–30) and generated an expanded sialome sublibrary with the human embryonic kidney (HEK) 293 for dissection of Siglec binding properties. First, combinatorial gene knockout (KO) was used to delete distinct subsets of sialyltransferase isoenzymes or all endogenous sialylation capacity. Second, using targeted gene knock-in (KI), individual sialyltransferase isoenzymes were introduced in the absence of other isoenzymes. Finally, we introduced selected sulfotransferase isoenzymes to explore cross-talk between sialylation and sulfation. To specifically address the influence of clustered O-glycan presentation for Siglec binding, we introduced a large panel of reporter constructs designed to display human O-glycodomains derived from mucins and mucin-like O-glycoproteins with different densities and patterns of O-glycans. The cell-based sialome array reproduced previous results for binding specificities for Siglec-2 (CD22) and Siglec-9 and led to insight into the binding specificities of Siglec-4/7/15 for distinct GalNAc-type O-glycans and their presentation on O-mucin–like glycoproteins. Finally, we demonstrate that Siglec-3/7/8/15 have preferential binding to sulfated sialoglycans yet have different specificities for underlying glycoconjugate structures. We further discovered the 6′-Su-SLacNAc (Neu5Acα2-3[6-O-sulfo]Galβ1-4GlcNAc) epitope on N-glycans and glycolipids as the ligand for Siglec-3/CD33 as well as Siglec-8. In summary, the cell-based display of the human sialome enables dissection of the Siglec interactome in the natural context of a human cell and provides the biosynthetic and genetic basis for the identified ligands.     

Imaging-guided percutaneous biopsy of focal splenic lesions: update on safety and effectiveness

OBJECTIVE: The purpose of this study is to determine the safety and effectiveness of percutaneous imaging-guided biopsy in the diagnosis of focal splenic lesions. MATERIALS AND METHODS: From May 1995 to November 1997, 20 imaging-guided biopsies of focal splenic lesions were performed in 18 patients, including seven patients with a prior diagnosis of extrasplenic malignancy (breast cancer, n = 3; lymphoma, n = 2; ovarian cancer, n = 1; and osteogenic sarcoma, n = 1), three immunosuppressed patients (cause of immunosuppression: AIDS, n = 1; liver transplantation, n = 1; and bone marrow transplantation, n = 1), two patients with anemia, one patient with a recent history of IV drug abuse, and five patients with incidentally discovered splenic lesions. Biopsies were performed with an 18-gauge (n = 1), a 20-gauge (n = 8), or a 22-gauge (n = 14) self-aspirating needle or an 18-gauge cutting needle (n = 1). Biopsies were considered successful if a specific diagnosis of benign or malignant disease was made. RESULTS: A specific diagnosis was made in 16 (88.9%) of 18 patients, and no complications occurred. Malignancy was diagnosed in six patients including three patients with lymphoma. Benign conditions were diagnosed in 10 patients: a cyst in two patients; hamartoma in one; lipogranuloma in one; infarct in one; and infection in four, including one case each of Candida albicans, Pneumocystis carinii, Mycobacterium tuberculosis, and mixed flora. The tenth benign diagnosis was a pseudotumor of the spleen related to a bulbous tail of the pancreas that was inseparable from the splenic hilum. Biopsy did not establish a diagnosis in one patient with lymphoma and in one patient with presumed splenic candidiasis. A mean of 1.5 needle passes was made per biopsy. CONCLUSION: Imaging-guided splenic biopsy is a safe technique that provides a specific diagnosis in most patients with focal splenic lesions.